The document discusses techniques for DNA sequencing, including early methods developed in the 1970s by Maxam and Gilbert as well as Sanger. It provides details on how both methods work, such as using specific chemical or enzymatic reactions to generate labeled DNA fragments of different lengths corresponding to nucleotide positions in the sequence. The document also describes how these methods were later automated, using fluorescent tags on dideoxynucleotides and capillary electrophoresis to simultaneously sequence multiple samples in a single gel. This allowed rapid determination of thousands of nucleotides and enabled large genome sequencing projects such as the Human Genome Project.

1. DNA Sequencing
Techniques
Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar

2. •In its capacity as a repository of information, a DNA molecule’s most important
property is its nucleotide sequence.
•Until the late 1970s, determining the sequence of a nucleic acid containing even
five or ten nucleotides was difficult and very laborious.
•The development of two new techniques in 1977, one by Alan Maxam and Walter
Gilbert and the other by Frederick Sanger, has made possible the sequencing of
ever larger DNA molecules with an ease unimagined just a few decades ago.

3. •In both Sanger and Maxam-Gilbert sequencing, the general principle is to reduce the
DNA to four sets of labeled fragments.
•The reaction producing each set is base-specific, so the lengths of the fragments
correspond to positions in the DNA sequence where a certain base occurs.
• For example, for an oligonucleotide with the sequence pAATCGACT, labeled at the 5’
end (the left end), a reaction that breaks the DNA after each C residue will
generate two labeled fragments: a four-nucleotide and a seven-nucleotide fragment;
• a reaction that breaks the DNA after each G will produce only one labeled, five
nucleotide fragment.
•Because the fragments are radioactively labeled at their 5’ ends, only the fragment
to the 5’ side of the break is visualized. The fragment sizes correspond to the relative
positions of C and G residues in the sequence.
•When the sets of fragments corresponding to each of the four bases are
electrophoretically separated side by side, they produce a ladder of bands from
which the sequence can be read directly.

4. Maxam Gilbert’s chemical degradation methods
•Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan
Maxam and Walter Gilbert in 1976–1977.
• This method is based on nucleobase-specific partial chemical modification of
DNA and subsequent cleavage of the DNA backbone at sites adjacent to the
modified nucleotides
•Maxam–Gilbert sequencing was the first widely adopted method for DNA
sequencing, along with the Sanger dideoxy method.

5. In this method, which is illustrated in Figure, following
steps are involved :
(i) Lebel the 3' ends of DNA with 32P.
(ii) Separate two strands, both lebel led at 3' ends,
(iii) Divide the mixture in four samples, each treated with
a different reagent having the property of destroying
either only G, or only C, or A and G, or T and C. The
concentration of reagent is so adjusted that 50% of target
base is destroyed, so that fragments of different sizes
having 32P are produced,
(iv) Electrophorese each of the four samples in four
different lanes of the gel.
(v) Autoradiograph the gel and determine the sequence
from positions of bands in four lanes.

6. DNA sequencing by the Sanger
method
• This method makes use of the
mechanism of DNA synthesis by DNA
polymerase.
(a)DNA polymerases require both a
primer(a short oligonucleotide
strand),to which nucleotides are
added, and a template strand to
guide selection of each new
nucleotide.
In cells, the 3‘-hydroxyl group of the
primer reacts with an incoming
deoxynucleoside triphosphate
(dNTP)to form a new phosphodiester
bond.
(b) The Sanger sequencing
procedure uses dideoxynucleoside
triphosphate
(ddNTP) analogs to interrupt DNA
synthesis.
(The Sanger method is also known
as the dideoxy method.)
When a ddNTP is inserted in place
of a dNTP, strand elongation is
halted after the analog is added,
because it lacks the 3'-hydroxyl
group needed for the next step

7. (c)The DNA to be sequenced is
used as the template strand,
and a short primer,
radioactively or fluorescently
labeled, is annealed to it. By
addition of small amounts of a
single ddNTP, for example
ddCTP, to another wise normal
reaction system, the
synthesized strands will be
prematurely terminated at
some locations where dC
normally occurs. Given the
excess of dCTP over ddCTP the
chance that the analog will be
incorporated whenever a dC is
to be added is small.
However, ddCTP is present in
sufficient amounts to ensure
that each new strand has a
high probability of acquiring
atleast one ddC at somepoint
during synthesis. The result is
a solution containing a
mixture of labeled fragments,
each ending with a C residue.
Each C residue in the
sequence generates a set of
fragments of a particular
length, such that the different
sized fragments, separated by
electrophoresis, reveal the
location of C residues.
This procedure is repeated
separately for each of the
four ddNTPs, and the
sequence can be read directly
from an autoradiogram of the
gel. Because shorter DNA
fragments migrate faster, the
fragments near the bottom of
the gel represent the
nucleotide positions closest
to the primer(the5' end), and
the sequence is read (in the
5' to 3' direction) from
bottom to top. Note that the
sequence obtained is
that of the strand
complementary to the strand
being analyzed.

8. Automated DNA sequencing
•DNA sequencing is readily automated by a variation of Sanger’s sequencing
method in which the dideoxynucleotides used for each reaction are labeled
with a differently colored fluorescent tag.
•This technology allows DNA sequences containing thousands of nucleotides to be
determined in a few hours.
•Entire genomes of many organisms have now been sequenced, and many very
large DNA sequencing projects are in progress.
• Perhaps the most ambitious of these is the Human Genome Project, in
which researchers have sequenced all 3.2 billion base pairs of the DNA in a human
cell.

9. Strategy for automating DNA
sequencing reactions
•Each dideoxynucleotide used in the
Sanger method can be linked to
a fluorescent molecule that gives all
the fragments terminating in that
nucleotide a particular color.
•All four labeled ddNTPs are added
to a single tube.

10. •The resulting colored DNA fragments are then
separated by size in a single electrophoretic gel
contained in a capillary tube.
•All fragments of a given length migrate
through the capillary gel in a single peak, and
the color associated with each peak is
detected using a laser beam.
•The DNA sequence is read by determining the
sequence of colors in the peaks as they pass
the detector.
•This information is fed directly to a computer,
which determines the sequence.

11. Thank You